Arabinoxylans (AX) are an important constituent of the cell walls of cereal grains. AX are carbohydrates consisting of a backbone of β-1,4-linked D-xylopyranoside (xylose) units that are either unsubstituted, monosubstituted with a single α-L-arabinofuranoside (arabinose) at either C—(O)-2 or C—(O)-3, or disubstituted with single α-L-arabinofuranoside units at C—(O)-2 and C—(O)-3 (Izydorczyk and Biliaderis, 1995; Andersson and Aman, 2001;). The pentoses xylose and arabinose are thus the main building blocks of AX, and AX are therefore also called pentosans. Less abundant substituents attached to the C—(O)-2 position of the xylose units can be glucuronic acid, 4-O-methyl glucuronic acid, or short oligomers consisting of L-arabinose, D-xylose, D-galactose, D-glucose and/or uronic acids, while acetyl groups can be linked to the C—(O)-2 and/or C—(O)-3 position of the xylose units. Hydroxycinnamic acids, mainly ferulic acid, and to a lesser extent dehydrodiferulic acid, p-coumaric acid, and sinapic acid, are present as substituents as well, and they are generally linked to the C—(O)-5 position of terminal arabinose units (Izydorczyk and Biliaderis, 1995; Andersson and Aman, 2001).
Apart from their common structural feature of a xylose backbone substituted with arabinose, cereal grain AX constitute a very heterogenous group of carbohydrates. Large variations in degree of arabinose substitution and nature and frequency of the less abundant substituents have been observed, not only when comparing different cereal species but also when comparing different tissues within a single cereal species (Antoine et al., 2003; Barron et al., 2007). It is generally conceded that cereal AX have a degree of polymerisation (DP) between 1,500 and 15,000, although it is difficult to determine their exact molecular weight because of the partial degradation that typically takes place during their extraction and purification (Broekaert et al., 2011).
Overall, cereal grains contain between 5 and 10% of AX. However, AX are not evenly distributed over the different tissues of the cereal grain. Particularly the outer layers of the cereal grain are rich in AX, explaining the common practice to first physically fractionate the cereal grain to obtain a fraction enriched in the outer layers (commonly named “bran”) to serve as raw material for the preparation of AX derived products. However, the major part of the AX from the outer layers of the cereal grain are water-unextractable and cannot be isolated in their native state because they are intimately associated with other cell wall materials by both covalent and non-covalent interactions. As a result, isolation of a substantial fraction of the AX from bran often involves at least partial hydrolytic depolymerisation of the AX. Furthermore, this depolymerisation of the AX is mostly also desirable from an application point of view. Indeed, the high viscosity that comes with the high molecular weight of AX is often not desired for many applications of AX.
Depending on their DP, solubilised AX depolymerisation products find their way in different applications. For instance, arabinoxylan-oligosaccharides (AXOS) have recently been shown to exert prebiotic properties (Cloetens et al., 2008; Courtin et al., 2008; Van Craeyveld et al., 2008; Broekaert et al., 2011). AXOS containing preparations therefore have a wide potential as ingredient in food, beverage and feed applications. AX can also be used as an ingredient for cosmetics. The pentoses xylose and arabinose could be used for fermentation, for instance for the production of ethanol, if yeast strains are engineered such that they can utilise and ferment xylose and arabinose (Hahn-Hägerdal et al., 2007). The pentose xylose can be used in applications in the pharmaceutical, cosmetic and food industry, and as a substrate for enzymatic or chemical conversion to xylitol, which can be used as a non-cariogenic sweetener. The pentose arabinose is widely used as an intermediate in the production of pharmaceuticals, such as nucleoside analogue antiviral agents.
Several methods have been described to prepare pentoses or pentose-based oligosaccharides from cereal bran.
AX depolymerisation products can be prepared from cereal bran by aqueous extraction in the presence of endoxylanase enzymes (WO 02/067698; WO 2006/027529; Maes et al., 2004; Swennen et al., 2006). These methods have the benefit of being based on gentle extraction conditions; however, recoveries of AX are typically limited due to the inaccessibility of an important part of the cereal bran AX to the enzymes.
Higher recoveries can be obtained with alkaline extraction conditions (U.S. Pat. No. 3,879,373; WO 98/31713). Extraction methods that combine alkali with peroxide (Maes and Delcour, 2001; Hollmann and Lindhauer, 2005) have been described as well. The main drawback of these methods is that the extraction step with high concentration of alkali is environment-unfriendly, and requires costly removal of the chemicals from the different product streams. Furthermore, the AX extracted with alkaline solutions have a high molecular weight and an additional endoxylanase treatment (Yamada et al., 1993; Beaugrand et al., 2004) is generally still required to obtain AX depolymerisation products with a desired molecular weight. Finally, the AX extracted with alkaline solutions are devoid of hydroxycinnamic acid substituents (Hollmann and Lindhauer, 2005), most likely due to saponification of the ester link. The lack of hydroxycinnamic acid substituents can be a drawback for the use of such preparations in food and cosmetics applications, since the hydroxycinnamic acid substituents confer desired antioxidant properties to the AX or AXOS (Ohta et al., 1997; Yuan et al., 2005; Vitaglione et al., 2008).
Higher recoveries can also be obtained with acidic extraction conditions. Different extraction methods with acid solutions at high temperature have been described (Sanjust et al., 2004; Palmarola-Adrados et al., 2005; WO 2010/088744). However, these methods are generally more suited to produce pentoses rather than pentose-based oligosaccharides, due to extensive acid-catalysed hydrolysis of the polysaccharide links.
Finally, high recoveries can be obtained in aqueous solutions using a hydrothermal treatment at high pressure and high temperature leading to autohydrolysis of AX (Garrote et al., 2002; Kabel et al., 2002; Carvalheiro et al., 2004; Rose and Inglett, 2010). A potential drawback of this treatment is the release of substantial amounts of free monosaccharides which may not always be desired. Furthermore, the conditions leading to autohydrolysis of AX, also lead to the formation of pentose degradation products such as furfural.
Bran is conventionally produced as a by-product of milling of cereal grain using a roller mill. In a roller mill, the cereal grain is ground by the action of pairs of rolls rotating in opposite direction and sieves are used for separation of ground cereal grain fractions. Bran produced by roller milling comprises a range of different tissues, including pericarp, seed coat, nucellar epidermis, aleurone as well as minor parts of the starchy endosperm (Delcour and Hoseney, 2010). It is known that these different tissues not only do have different contents of AX, the AX from the different tissues also differ in structure and hence in accessibility by endoxylanases. More particularly, the lesser arabinose substituted AX from the aleurone and nucellar epidermis appear to be well solubilised by the action of endoxylanases while this is much less the case for the more complex AX from the pericarp (Benamrouche et al., 2002; Ordaz-Ortiz at al., 2005; Van Craeyveld et al., 2010).
Another method for producing cereal bran is debranning. Debranning is the controlled removal of peripheral layers of cereal grain while leaving the remaining cereal grain substantially intact. Debranning can be done by friction (peeling), i.e. the rubbing of cereal grains against each other, or by abrasion (pearling), i.e. the rubbing of cereal grains against an abrasive surface, or by a combination of both (Hemery et al., 2007).
It is an object of the present invention to provide more cost efficient methods for the extraction and isolation of soluble AX products from cereals than the methods known from the prior art. The higher cost efficiency is based on alternative methods to prepare bran from cereal grain that result in higher yields from bran in the preparation of the soluble AX products. It was unexpectedly found that, while higher yields were not obtained with bran obtained by debranning of cereal grain compared to bran obtained by roller milling, higher yields were obtained with bran prepared by roller milling of partially debranned cereal grain.